Abstract The goal of the proposed project is to study the changes in the serum virome of transfusion recipients as it relates to the development of post-transfusion hepatitis (PTH). In addition to utilizing important archived NHLBI biorepository samples for identifying new viruses in the PTH subjects, our aim is also to generate an enormous amount of novel sequence data of known transfusion transmitted viruses, including hepatitis C, B and D viruses, and other common blood viruses like Torque teno viruses, GBV-C and herpesviruses. Recently, we identified a new flavivirus, Human hepegivirus (HHpgV-1), in serum samples of transfusion recipients. Simultaneously, the HHPgV-1 infection was also reported in serum samples of HCV infected patients in California and more recently from HCV infected people in Europe. Unbiased characterization of all viruses in these samples will fill the knowledge gap that remained after diagnostic identification of HCV in the PTH subjects. Towards this, we will study all viruses, their genotypes and variants for changes in their composition over time in the subjects of two landmark PTH studies, Natural History Study of Non-A, Non-B Post-Transfusion Hepatitis (NANB-TAH) and VA Cooperative Study of the Efficacy of Hepatitis Immune Serum Globulin for the Prevention or Modification of Post-Transfusion Hepatitis (VA2-TAH). We will use the most up-to-date virome analysis methodology and an improved sequencing library preparation method for comprehensive characterization of all viruses in these samples. We propose two specific aims: Aim-1 is to characterize all new and known virus populations in pre- and post-transfusion samples of VA2-TAH and NANB-TAH subjects. Aim-2 is to study the evolution and nature of infections of transmitted virus populations. We will compare viruses and their variants in the serum samples of PTH subjects, matched donors and serially collected samples of recipients. We expect to determine the relationships between virome composition changes, viral diversity (genotypes/lineages) and elevated ALT levels in PTH subjects. Moreover, we will characterize the patterns of virus evolution and the population structures of transmitted viruses using various phylogenetic approaches. Our proposed comprehensive analysis of viromes in these samples will advance our knowledge of all transfusion transmitted viruses.